Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito 99% resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development prior to invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species.

The R7 family of RGS proteins (RGS6, -7, -9, -11) is characterized by the presence of three domains: DEP, GGL, and RGS. The RGS domain interacts with Galpha subunits and exhibits GAP activity. The GGL domain permanently associates with Gbeta5. The DEP domain interacts with the membrane anchoring protein, R7BP. Here we provide evidence for a novel interaction within this complex: between the DEP domain and Gbeta5. GST fusion of the RGS7 DEP domain (GST-R7DEP) binds to both native and recombinant Gbeta5-RGS7, recombinant Gbetagamma complexes, and monomeric Gbeta5 and Gbeta1 subunits. Co-immunoprecipitation and FRET assays supported the GST pull-down experiments. GST-R7DEP reduced FRET between CFP-Gbeta5 and YFP-RGS7, indicating that the DEP-Gbeta5 interaction is dynamic. In transfected cells, R7BP had no effect on the Gbeta5/RGS7 pull down by GST-R7DEP. The DEP domain of RGS9 did not bind to Gbeta5. Substitution of RGS7 Glu-73 and Asp-74 for the corresponding Ser and Gly residues (ED/SG mutation) of RGS9 diminished the DEP-Gbeta5 interaction. In the absence of R7BP both the wild-type RGS7 and the ED/SG mutant attenuated muscarinic M3 receptor-mediated Ca2+ mobilization. In the presence of R7BP, wild-type RGS7 lost this inhibitory activity, whereas the ED/SG mutant remained active. Taken together, our results are consistent with the following model. The Gbeta5-RGS7 molecule can exist in two conformations: "closed" and "open", when the DEP domain and Gbeta5 subunit either do or do not interact. The closed conformation appears to be less active with respect to its effect on Gq-mediated signaling than the open conformation.

Members of the RGS7 (R7) family and Gβ5 form obligate heterodimers that are expressed predominantly in the nervous system. R7-Gβ5 heterodimers are GTPase-activating proteins (GAPs) specific for Gi/o-class Gα subunits, which mediate phototransduction in retina and the action of many modulatory G protein-coupled receptors (GPCRs) in brain. Here we have focused on R7BP, a recently identified palmitoylated protein that can bind R7-Gβ5 complexes and is hypothesized to control the intracellular localization and function of the resultant heterotrimeric complexes. We show that: 1) R7-Gβ5 complexes are obligate binding partners for R7BP in brain because they coimmunoprecipitate and exhibit similar expression patterns. Furthermore, R7BP and R7 protein accumulation in vivo requires Gβ5. 2) Expression of R7BP in Neuro2A cells at levels approximating those in brain recruits endogenous RGS7-Gβ5 complexes to the plasma membrane. 3) R7BP immunoreactivity in brain concentrates in neuronal soma, dendrites, spines or unmyelinated axons, and is absent or low in glia, myelinated axons, or axon terminals. 4) RGS7-Gβ5-R7BP complexes in brain extracts associate inefficiently with detergent-resistant lipid raft fractions with or without G protein activation. 5) R7BP and Gβ5 protein levels are upregulated strikingly during the first 2-3 weeks of postnatal brain development. Accordingly, we suggest that R7-Gβ5-R7BP complexes could regulate signaling by modulatory Gi/o-coupled GPCRs in the developing and adult nervous systems.

Regulators of G protein signaling (RGS1) are a diverse family primarily known as GTPase-activating proteins (GAPs) for heterotrimeric G proteins. In addition to the RGS domain, which is responsible for GAP activity, most RGS proteins contain other distinct structural motifs. For example, members of the R7 family of RGS proteins contain a DEP, GGL and novel DHEX domain, and are obligatory dimers with the G protein beta subunit Gβ5. Here we show that the Gβ5-RGS7 complex can inhibit Ca2+ mobilization elicited by the muscarinic acetylcholine receptor type 3 (M3R), but not by other Gq-coupled receptors such as M1, M5, histamine H1 and GNRH receptors. Isolated DEP domain of RGS7 is sufficient for the inhibition of M3R signaling, whereas the deletion of the DEP domain renders Gβ5-RGS7 ineffective. Deletion of a portion of the 3rd intracellular loop allowed the receptor (M3R-short) to signal, but rendered it insensitive to the effect of Gβ5-RGS7. Accordingly, recombinant DEP domain bound in vitro to the GST-fused i3 loop of the M3R. These results identify a novel molecular mechanism that can impart receptor-subtype selectivity on signal transduction via Gq-coupled muscarinic receptors.